Targeted at learning about pathogenic processes of inflammatory eye diseases, this project focused in FY 2009 on populations of T-cells involved in these processes. We investigated two populations of recently identified T-helper cells, specifically producing IL-17 ("Th17" cells), or IL-9 ("Th9" cells). 1) Th17 Cells. Our studies in FY 2008 defined several basic features of Th17 cells with specificity against an ocular antigen. In those previous experiments, we generated lines of Th17 cells from naive CD4 cells transgenically expressing a T-cell receptor (TCR) specific against hen egg lysozyme (HEL). We activated these CD4 cells by their incubation with HEL, provided by antigen-presenting cells (APC), in the presence of a polarizing cytokine mixture of IL-6 and TGF-beta. These Th17 cell lines were pathogenic, inducing ocular inflammation in transgenic recipient mice that express HEL in their eyes. This study was extended in FY 2009 as described below. In these new studies we used another procedure to generate Th17 cells with specificity toward an ocular antigen. In this procedure, CD4 are activated by plate-bound antibodies against CD3 and CD28, in the presence of IL-6 and TGF-beta. We found that Th17 lines generated by this procedure are non-pathogenic when injected into recipients expressing HEL in their eyes: no pathological changes were detected in eyes of recipients after adoptively transferring as many as 5 million of these Th17 cells. These data thus show that by modifying the mode of activation, we obtained two phenotypes of Th17, one immunopathogenic the other is not. We then compared the two lines of Th17, activated by either HEL/APC or plate-bound antibodies, for the additional following parameters: (i) Effects of culture additives. The polarizing cytokines IL-6 and TGF-beta are essential for generation of the non-pathogenic Th17 lines for which activation is provided by plate-bound anti-CD3/CD28 antibodies. In contrast, addition of IL-6 and TGF-beta did not affect the pathogenicity of Th17 lines activated by HEL/APC. It is assumed that IL-23 produced by the APC determines the pathogenicity of the latter Th17 lines. (ii) Cytokine production. Both Th17 lines secreted IL-17, but cells activated by the plate-bound antibodies released approximately 5-fold higher levels of this cytokine than did HEL/APC-activated Th17 cells. In contrast, HEL/APC-activated cells secreted 50-fold higher levels of IL-22, a cytokine related to the immunopathogenic activity of Th17. (iii) Migration-related molecules. Th17 cells generated by activation with plate-bound antibodies also differed from those activated by HEL/APC in their expression profiles of chemokines, chemokine receptors and adhesion-related molecules. It is noteworthy that the profile of these molecules expressed by cells of the latter Th17 subtype enables more efficient invasion into non-lymphoid tissues, in line with the pathogenicity of these cells. 2) Antigen-Specific Th9 Cells. The generation in vitro of Th9 cell lines, specifically producing IL-9, was reported for the first time in 2008 (Nat. Immunol., 9:1341 and 9:1347). These lines also produced IL-10 and were generated by activating wild-type naive CD4 cells with antibodies against CD3 and CD28. These lines did not exhibit, however, any antigen specificity. We used our system of transgenic mice, described above, to generate and characterize for the first time antigen-specific Th9 lines. We generated these cell lines by incubating naive CD4 cells, transgenically expressing HEL-specific TCR, with HEL and APC, in the presence of the polarizing cytokines, IL-4 and TGF-beta. The Th9 lines we generated expressed high levels of IL-9 and IL-10, but only trace levels of IL-17 and interferon-gamma, thus verifying their high level of purity. Intracellular analysis of cytokine production by these line cells revealed a unique pattern of IL-9 expression: an early and rapid production of this cytokine, with as many as 45% of the line cells expressing IL-9 on day 3, followed by a sharp decline, to 1% IL-9 positive cells on day 6 of culture. In contrast, expression of intracellular IL-10 by the Th9 line cells increased gradually, with the highest levels of intracellular IL-10, of approximately 40%, found on day 6 in culture. The pattern of IL-10 production resembles that of other cytokines, such as IL-17 or interferon-gamma, in Th17 or Th1 cells, respectively, and thus, the difference we found between IL-9 and IL-10 production re-emphasizes the uniqueness of IL-9 expression pattern. Despite their specificity toward HEL, cells of the Th9 lines were non-pathogenic: no ocular pathology was induced in recipient mice expressing HEL in their eyes following injection of as many as 5 million activated cells of these Th9 lines.